| In this thesis, we reported flower bud differentiation rules and the role of endogenous factors regulation of flowering in Agapanthus praecox ssp. orientalis. We used Agapanthus praecox ssp. orientalis as the research object, and studied anatomy of floral buds development, embryology, flowering physiology, analysis of endogenous hormones, transcriptomics and proteomics during reproductive stage. The main results are as follow.1. Anatomy of floral buds development:Floral organs differentiation was comprised of 6 distinct stages including nought differentiation, inflorescence bud differentiation, floret primordia differentiation, tepal primordia differentiation, stamen primordia differentiation and pistil primordia differentiation.6 tepals differentiated almost simultaneously which cross arrange in space and appear in hexagonal distribution pattern.6 stamens differentiated inside the tepals at the same time. Finally,3 carpel primordia differentiated and formed syncarpous pistil. The whole process of floral bud differentiation took approximately 40 days with the first 3 stages developing more slowly than the later 3 stages. Morphology and color of the anther underwent obvious changes during the period between stamen primordia differentiation and anther maturation. Microspores also underwent significant development during this same interval.2. Embryology:The anther wall, whose development is of dicotyledonous type, consists of four layers:the epidermis, the endothecium, one or two middle layers, and the secretory tapetum. Fibrous thickenings are developed in the endothecium when shed. Successive cytokinesis during microsporogenesis results in a decussate tetrad of microspores. Ellipsoidal pollen grains are 2-celled when anthers dehisce, with monosulcate extending to polar area; pollen ornamentation is pitted and reticulate. Ovary is superior and trilocular, with axial placenta. The ovule is anatropous, bitegmic, and tenuinucellate. The archesporial cell below nucellus epidermis functions directly as the megasporocyte. Successive cytokinesis in the megasporocyte usually produces a T-shape tetrad, and the chalazal megaspore of the tetrad develops into a Polygonum-type embryo sac. The pollen tube enters the embryo sac and discharges two sperm 44-48 h after pollination. The sperm fuse with the egg cell and polar nuclei, forming zygote and primary endosperm nucleus, approximately 50 h after pollination. The zygote then enters a short period of dormancy. Seven days after pollination, the zygote starts division. The first division of the zygote is transversal. The embryo undergoes globular stage, rodshaped stage, and finally forms a monocotyledonous embryo. The suspensor cells are ephemeral and degenerate at the globular embryo stage. Endosperm cells contain massive starch grains as nutrition for embryo development. Embryogeny conforms to the Onagrad type. and endosperm formation is of the nuclear type; the whole process of embryogeny and endosperm development needs approximately 60 days in A. praecox ssp. orientalis. Dicotyledonous together with monocotyledonous forms of embryo morphogenesis in Agapanthus supports the concept of homo logy of monocots and dicot cotyledons.3. Flowering physiology:every physiological index increased significantly in the stages of vegetative phase and florescence. The content of RNA and the ratio of RNA/DNA present double peak curve in leaves against single peak curve in stem apex; the total starch content of leaves increased during the initial stages of floral bud differentiation and then decreased, while the starch content of stem apex kept decreasing from the initial stages of floral bud differentiation and increased significantly until the stage of florescence; the total soluble sugar content of leaves exhibited sustained low, while the content of soluble sugar of stem increased significantly during the stage of floral bud differentiation and then decreased, and increased again at the stage of floral bud development. In the whole process of flower development, the content of soluble sugar, starch and amylase of leaves always lower than that of stem apex.4. Analysis of endogenous hormones:11 kind of GA were detected in Agapanthus, and GA4 as bioactive gibberellic acid play an important role in regulation of flowering. GA4 contents were increased in stem apex at induction and floral buds development periods, the contents were 2.76 and 2.63 folds compared to vegetative period. Immunohistochemical analysis indicating that GA are mainly distribute in floret primordia and ovules. IAA contents changed most significantly in stem apex during floral buds development. In induced buds IAA contents increased by 581%contrast to vegetative buds. Strong IAA immunofluorescence signals were detected in floret primordia, floral organ primordia, tapetum, filament and ovules. So, IAA plays an important role in regulating floral buds development in Agapanthus. Zeatin as bioactive cytokinin plays an important role in regulation of flowering. Zeatin contents increased in process of flower buds differentiation and Zeatin mainly distributed in floret primordia and ovules. The contents of cis-ABA were decreased during flower bud development. Comprehensive analysis shows that GA, IAA, CTK promote flowering and ABA negative regulate flowering in Agapanthus. Higher ratio of IAA/ABA promote flowering. Lower ratio of GA3+4/IAA promotes floral organs differentiation and higher ratio of GA3+4/IAA increase scape elongation, higher ratio of (IAA+GA3+GA4+Z+iP)/cis-ABA play a decisive role on flowering.5. Transcriptomics:a total of 72 differentially expressed genes were screen out by used cDNA-AFLP technology. The main function of genes encoding proteins were classified into 12 categories of which the largest proportion is metabolic genes (22%). quantitative expression analysis were application on 33 genes with important regulatory functions. There are 23 genes up-regulated and 2 genes down-regulated in floral buds during reproductive stage; 8 genes up- regulated only in induced buds. TIR1 is a major receptor protein of IAA, the mRNA levels of TIR1 up-regulated in the whole process of floral buds development. GAI is a negative regulation transcription factor of gibberellin signal, the mRNA levels of GAI up-regulated in induced buds and down-regulated in reproductive buds. These phenomenons indicating that IAA promote flowering, lower level of GA control the flowering and higher level of GA promote floral organs development.6. Proteomics:a total of 72 differentially expressed proteins were separated by two-dimensional gel electrophoresis. Among these proteins, there are 26 proteins up-regulated and 15 proteins down-regulated in the whole process of reproductive stage, and 21 proteins exclusively up-regulated in induced buds. These proteins classified by function into 12 categories of which the largest proportion is metabolic proteins (35%). Comprehensive analysis shows that sugar synthesis and metabolic pathway, H2O2 and ethylene regulation pathway may be important endogenous regulatory factors of flowering in Agapanthus.7. Bioinformatics analysis:Differentially expressed genes and proteins involved in photosynthetic carbon fixation pathway and Glycolysis/Gluconeogenesis pathway. After a comprehensive analysis indicating that important endogenous factors regulating Agapanthus flowering are mainly including:IAA signaling pathway, GA signaling pathway, ethylene signaling pathway, H2O2 regulatory pathway and sugar synthesis and metabolic pathway.
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